2. Introduction
A DNA library is a collection of DNA fragments that
have been cloned into vectors so that researchers can
identify and isolate the DNA fragments that interest
them for further study.
There are basically two kinds of libraries: genomic
DNA and cDNA libraries.
Genomic DNA libraries contain large fragments of DNA
in either bacteriophages or bacterial or P1-derived
artificial chromosomes (BACs and. PACs). cDNA
libraries are made with cloned, reverse-transcribed
mRNA, and therefore lack DNA sequences
corresponding to genomic regions that are not
expressed, such as introns and 5′ and 3′ noncoding
regions.
cDNA libraries generally contain much smaller
3. Library Screening
The identification of specific clone from a DNA
library can be carried out by exploiting either:
1. the sequence of the clone or
2. the structure/function of its expressed product.
Former type can be applied to genomic or cDNA
libraries and nucleic acid hybridization is done
with the help of probes or primers.
Screening the product of a clone is applied only
to expression libraries where the DNA fragment is
expressed to yield proteins and the product is
recognized by Ab/ligand.
4. Sequence dependent Screening
Screening by Hybridization
Nucleic acid hybridization is most commonly used
method and it is rapid.
Developed by Grunstein and Hogness (1975).
This method is widely used in isolating
Recombinant phage particles.
Thus, hybridization has potential to isolate any
sequence if probe is available.
Variation of this method devised by Benton is
called Plaque lift method.
5.
6. PCR Screening
The PCR is widely used to isolate specific DNA
sequences from uncloned genomic DNA and now
it has been a useful technique for library
screening.
This method was first demonstrated by takumi
and Lodish in 1994.
The molecular weight of known members of the
family can be predicted and novel mRNAs may
give rise to amplification products.
It is more stringent since three oligonucleotides
(the two PCR primers, and the hybridization
probe) are required to give a true positive signal.
7.
8. South-western and North-western
Screening
A plaque lift is carried out to transfer a print of the
library onto nitrocellulose membrane.
Here screening is carried out by incubating radio
labelled double stranded DNA oligonucleotide
probe containing recognition sequence for the
DNA-binding protein.
Combines the principles of southern and western
blots. It has been particularly successful in the
isolation of clones expressing cDNA sequences.
Alternatively ligands can be used to identify
polypeptides that specifically bind certain
molecules.